The glandular kallikreins are a subgroup of serine proteases which are involved in the post-translational processing of specific polypeptide precursors to their biologically active forms. The rodent kallikrein gene family consists of at least 25 genes. However, the human kallikrein gene family is much smaller, consisting of three members: prostate-specific antigen, human glandular kallikrein, and pancreatic/renal kallikrein. See J. A. Clements, Endocr. Rev., 10, 393 (1989) and T. M. Chu et al. (U.S. Pat. No. 4,446,122). A common nomenclature for these members of the tissue (glandular) kallikrein gene families was recently adopted by T. Berg et al., in Recent Progress on Kinins: Biochemistry and Molecular Biology of the Kallikrein-Dinin System. Aqents and Actions Supplements, Vol. I, H. Fritz et al., eds., Birkhauser Verlag, Basel (1992), and is given on Table I, below.
TABLE I __________________________________________________________________________ The Human Tissue Kallikrein Gene Family (approved species designation: HSA) New Previous Designa- Designa- New Protein tion tions mRNA/cDNA Protein Designation __________________________________________________________________________ hKLK1 KLK1.sup.1,2 .lambda.HK1.sup.4 and tissue kallikrein.sup.6,14 hK1 hRKALL.sup.3 phKK25.sup.5 (renal/pancreas/ cDNAs salivary) hKLK2 KLK2.sup.7, prostate-specific hK2 hGK-1.sup.8, glandular kallikrein.sup.15 hGK-3.sup.5 hKLK3 PSA.sup.9, .lambda.HPSA-1.sup.11 PSA.sup.13 (prostate-specific hK3 PA.sup.10, and PSA.sup.12 antigen) APS.sup.1,2 cDNAs __________________________________________________________________________ .sup.1 G.R. Sutherland et al., Cytogenet. Cell Genet., 48, 205 (1988). .sup.2 M.M. LeBeau et al., Cytogenet. Cell Genet., 51, 338 (1989). .sup.3 B.A. Evans et al., Biochemistry, 27, 3124 (1988). .sup.4 A.R. Baker et al., DNA, 4, 445 (1985). .sup.5 D. Fukusima et al., Biochemistry, 24, 8037 (1985). .sup.6 F. Lottsperch et al., HoppeScyler's Z. Physiol. Chem., 360, 1947 (1979). .sup.7 H.H. Ropers et al., Cytogenet. Cell Genet., 55, 218 (1990). .sup.8 L.J. Schedlich et al., DNA, 6, 429 (1987). .sup.9 M. Digby et al., Nuc. Acids Res., 17, 2137 (1989). .sup.10 P.H.J. Riegman et al., Biochem. Biophys. Res. Comm., 159, 95 (1989). .sup.11 A. Lundwal et al., FEBS Lett., 214, 317 (1987). .sup.12 P. Schulz et al., Nuc. Acids Res., 16, 6226 (1988). .sup.13 K.W.K. Watt et al., PNAS USA, 83, 3166 (1986). .sup.14 H.S. Lu et al., Int. J. Peptide Protein Res., 33, 237 (1989). .sup.15 C.Y.F. Young et al., Biochemistry, 818, (1992).
Amino acid sequences deduced by L. J. Schedlich et al., DNA, 6, 429 (1987) and B. J. Morris, Clin. Exp. Pharmacol. Physiolo., 16, 345 (1989) indicate that hK2 may be a trypsin-like serine protease, whereas hK3 (PSA) is a chymotrypsin-like serine protease. Therefore, these two peptides may have different physiological functions.
Although the cDNA and genomic sequences for hK2 have been described, the functional hK2 protein has not yet been isolated and characterized from prostate tissues. The DNA sequence homology between hKLK2 and hKLK3 (exon regions) is 80%, whereas the homology between hKLK2 and hKLK1 is 65%. The deduced amino acid sequence homology of hK2 is greater with respect to hK3 and lower with respect to hK1; being 78% and 57%, respectively.
The similarities of gene structure and deduced amino acid sequences of these human kallikreins suggest that their evolution may involve the same ancestral gene. Moreover, as reported by Morris, cited supra.; P. Chapdelaine, FEBS Lett., 236, 205 (1988); and Young, Biochemistry, 818, (1992), both hK2 and hK3 are expressed only in the human prostate, while expression of hK1 is limited to the pancreas, submandibular gland, kidney, and other nonprostate tissues. The putative sequence of a polypeptide reported to correspond to human urinary kallikrein was disclosed by Amgen (EPA 297,913).
Interestingly, the hK2 gene is located about 12 kbp downstream from the hK3 gene in a head-to-tail fashion on chromosome 19. See P. H. Riegman et al., FEBS Lett., 247, 123 (1989). Thus, the relationship between hK2 and hK3 gene expression is very intriguing, especially with respect to their evolution and functional properties.
Tremendous interest has been generated in hK3 (PSA) because of the important role it plays as a marker to detect and to monitor the therapy of prostate carcinoma. Its usefulness as a marker is based on the elevated serum concentration of circulating hK3 proteins which are frequently associated with prostatic cancer. The serum concentration of hK3 has been found to be proportional to the cancer mass in untreated patients, but is also proportional to the volume of hyperplastic tissue in patients with benign prostatic hyperplasia (BPH). The serum levels of hK3 become reduced following prostate cancer therapy.
Currently, the Mayo Laboratory assays over 60,000 specimens for hK3 levels annually. Therefore, the high degree of sequence homology of hK2 with hK3 suggests that the levels both proteins may be useful in the diagnosis of prostate cancer. For example, the antibodies developed against hK3 now used in these assays could theoretically also recognize hK2, because of mutual contamination in the antigenic preparations used to develop the anti-hK3 antibodies, or because the above-mentioned structural similarities of the two proteins would yield cross-reacting antibodies. If elevated serum hK2 levels are not indicative of prostate cancer, then detection of hK2 by anti-hK3 antibodies could be responsible for the substantial percentage of false positive results which are observed in current hK3 assays. On the other hand, if circulating hK2 levels are also elevated above baseline levels in prostate cancer patients, detection of hK2 by hK2-specific antibodies would provide an alternative, confirmatory assay for prostate cancer.
However, despite the information which can be ascertained about hK2 from the DNA sequence and the cDNA probes, very little is known about the hK2 protein itself. The reason for this is that the protein has not been purified and characterized, and no method exists for measuring the protein in either prostate plasma, seminal plasma or in blood serum.
Therefore, a need exists for antibodies to hK2 (hGK-1) which do not cross-react with hK3 (PSA). A further need exists for an assay to determine the presence and/or level of hK2 in a physiological sample, without detecting hK3, or conversely, for an assay that can detect the presence and/or level of hK3 in a sample which is unaffected by the presence of hK2 in the same sample. A further need exists for improved and/or alternative methods to detect, stage and follow the course of prostate cancer.